Molecular and Functional Identification of the Na⁺/H⁺ Exchange Isoforms NHE1 and NHE3 in Isolated Bovine Articular Chondrocytes

Abstract

Cartilage matrix turnover is sensitive to changes in intracellular pH (pH_i) and previous studies have shown that articular chondrocytes regulate pH_i predominantly using an amiloride-sensitive Na⁺ / H⁺ exchanger (NHE) with hallmark properties of the housekeeper isoform NHE1. Immunoblotting studies have, however, demonstrated that bovine chondrocytes express both the NHE1 and NHE3 isoforms of Na⁺ / H⁺ exchange. In the present study the effect of exposure to serum on acid extrusion from chondrocytes has been studied. The pH-sensitive fluoroprobe BCECF was used to measure pH_i in isolated bovine articular chondrocytes, and proton equivalent membrane transporters were characterised for cells isolated in the absence and presence of 5% fetal bovine serum (FBS). The contribution of NHE isoforms to acid extrusion, following ammonium-induced acidification, was assessed using a combination of ion substitution and the specific NHE1 inhibitor HOE694. While Na⁺ -dependent acid extrusion was entirely inhibited by HOE694 in FBS untreated cells, the operation of both NHE1 and NHE3 was detected in cells exposed to FBS. In parallel, RT-PCR and immunohistochemistry experiments demonstrated both NHE1 and NHE3 mRNA and expression of both proteins respectively. While serum growth factors are virtually excluded from healthy cartilage they could permeate a damaged matrix. The regulatory characteristics of NHE3 are distinct from NHE1 so that an altered pattern of response to components of mechanical stress such as hyperosmolarity may be associated with increased access of growth factors in diseased tissue.

Modulation of H⁺ Transport Mechanisms by Interleukin-1 in Isolated Bovine Articular Chondrocytes

“…The NHE response to growth factors and cytokines commonly reflects the activation of this protein by direct phosphorylation, Ca 2+ -calmodulin complex binding or altered interactions with regulatory proteins, and the rapidity of our response suggests similar events are occurring here. In chondrocytes, growth factors upregulate the activity of the NHE1 isoform, as well as inducing expression of NHE3 [13]. Here, however, the observation that NHE-1 specific HOE694, amiloride and removal of Na + ions all inhibited acid extrusion to the same degree indicates that the enhanced NHE activity is solely mediated by NHE1.…”

“…In contrast, ADAMTS4 activity is controlled largely by posttranscriptional activation involving furin and MT4-MMP. [13][14][15][16]. In mammalian chondrocytes, HCO 3 --dependent membrane transport processes do not operate to a great extent, probably because the expression and activity of carbonic anhydrase is low [17].…”

Cell Confluence-induced Activation of Signal Transducer and Activator of Transcription-3 (Stat3) Triggers Epithelial Dome Formation via Augmentation of Sodium Hydrogen Exchanger-3 (NHE3) Expression

“…Confluent cells were incubated with each inhibitor at different concentrations until the dome appeared in control cells. S3226 was kindly provided by Dr. Robert J. Wilkins (33). Oncostatin M was purchased from PeproTech EC Ltd (London, UK).…”

Evolutionary origins of eukaryotic sodium/proton exchangers

“…Fish were the first vertebrates; thus these exchangers may be important for bone formation or resorption. Preliminary studies in osteoclasts showed a role for Na ϩ /H ϩ exchange in H ϩ extrusion linked to bone resorption (92,132) and in pH regulation of chondrocytes (141,145). Fish were the first species to have a cardiovascular system responsible for respiratory exchange (unlike invertebrate hearts that just circulated nutrients), and although some gastropods have gills, fish species were the first to have evolved advanced gills for respiratory exchange (which evolved into lungs in amphibians and more complex species).…”